Project Summary The RAS family of oncogenes are mutated in 25-30% of human cancers and regulate cell proliferation, cell survival, metastasis and evasion of apoptosis. The most frequent sites of mutations in RAS proteins are residues 12, 13 and 61 and X-ray crystallographic studies have suggested that these mutations result in a loss of their GTPase activity, thus keeping the mutant proteins in constitutively active GTP-bound states. A major consequence of RAS mutation is the constitutive activation of multiple signaling pathways, including the MEK/ERK and PI3K/AKT/mTOR pathways. Hence, targeting RAS-driven signaling pathways constitutes an important approach to treat these tumors. Unfortunately, the development of RAS inhibitors has been challenging due to the lack of well-defined pockets and cavities on the RAS surface. Here, we describe an important breakthrough to inhibit RAS-signaling pathways. It is now well established that RAS transmits growth signals through interaction with a large number of effector proteins involving the RAS-binding domains (RBDs) of these effectors. To disrupt this interaction, we undertook a novel strategy of developing small molecule inhibitors that bind to the RBDs of RAS-effector proteins and disable their interaction with RAS thereby blocking RAS signaling. This strategy led to the identification and characterization of two compounds, rigosertib, and 015040, both of which bind to RBDs of multiple RAS effectors with high affinity and block their interaction with RAS, thereby inhibiting multiple RAS-mediated signaling pathways. Rigosertib is orally bio- available with an excellent safety profile and is currently in Phase III clinical trials for the treatment of myelodysplastic syndrome. It is our hypothesis that inhibition of RAS-Effector interactions by RAS mimetics such as rigosertib and 015040 will inhibit RAS-activated signaling pathways resulting in an inhibition of RAS- mutant tumor growth. To test this hypothesis we propose to use RAS-mutant colorectal cancer model since colorectal cancer is the second leading cause of cancer-associated deaths in USA and world-wide. KRAS is mutated in approximately 45% of CRCs and currently there is no targeted therapy for this form of cancer. In this application, we propose to examine the effects of rigosertib and 015040 on the growth of CRCs using genetically engineered (GEMM) and patient-derived Human Xenograft (PDX) mouse models. We will also use the newly developed Multiplexed-kinase Inhibitor Beads (MIB) and ?Cancer Toolkit gain-of-function? technologies to determine whether colorectal cancer cells develop resistance to RAS-mimetics upon prolonged exposure and to determine the nature of kinases that might be the root cause of such resistance. If our proposed studies show that rigosertib and 015040 show good efficacy in CRCs that harbor KRAS mutations, our goal is to initiate clinical trials to determine their efficacy in CRC patients starting with rigosertib.